Highly Transformable Elite Inbred Line-PHWWE

ABSTRACT

A novel inbred maize line designated PHWWE and seed, plants and plant parts thereof. Methods for producing a maize plant that comprise crossing inbred maize line PHWWE with another maize plant. Methods for producing a maize plant containing in its genetic material one or more traits introgressed into PHWWE through backcross conversion and/or transformation, and to the maize seed, plant and plant part produced thereby. Hybrid maize seed, plant or plant part produced by crossing the inbred line PHWWE or a trait conversion of PHWWE with another maize line. Inbred maize lines derived from maize line PHWWE, methods for producing other inbred maize lines derived from inbred maize line PHWWE and the inbred maize lines and their parts derived by the use of those methods.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding and maizetransformation.

BACKGROUND OF THE INVENTION

Transformation of elite maize inbreds is an important technology fordeveloping maize inbreds and hybrids with improved agronomic traits.Work by Armstrong and others (D. D. Songstad, W. L. Petersen, C. L.Armstrong American Journal of Botany, Vol. 79, pp. 761-764, 1992) showedthat it was possible to interbreed a more culturable, agronomically poormaize line (A188) with an agronomically desirable, less transformableline (B73) to produce a novel line, Hi-II, with increased culturabilityand regeneration. Hi-II maize has been used for maize transformation fora number of years because of its high transformability and goodculturability, but Hi-II is a hybrid. Non-homozygous plants used indeveloping transgenic traits are problematic. It is easier to determinethe effects of a transgene when a uniform, homozygous, background isused in transgene development. Another disadvantage of using Hi-II intransformation is that it does not have the quality genetics that arepresent in current elite inbreds. When developing a transgenic productthe transgene is moved into an elite background through crosspollination. After the initial cross, backcrossing is used to remove asmuch of the Hi-II deleterious genome as possible. This is a laborintensive and time consuming process. It would therefore be beneficialto have a homozygous maize variety that has an elite genotype while alsomaintaining high transformability and good response in culture.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel inbred maize line,designated PHWWE and processes for making PHWWE. This invention relatesto seed of the inbred maize line PHWWE, to the plants of inbred maizeline PHWWE, to plant parts of inbred maize line PHWWE, and to processesfor making a maize plant that comprise crossing inbred maize line PHWWEwith another maize plant. This invention also relates to processes formaking a maize plant containing in its genetic material one or moretraits introgressed into PHWWE through backcross conversion and/ortransformation, and to the maize seed, plant and plant part produced bysuch introgression. This invention further relates to a hybrid maizeseed, plant or plant part produced by crossing the maize line PHWWE oran introgressed trait conversion of PHWWE with another maize line. Thisinvention also relates to inbred maize lines derived from maize linePHWWE to processes for making other inbred and doubled haploid maizelines derived from the PHWWE maize line and to the inbred maize linesand their parts derived by the use of those processes.

DEFINITIONS

ALLELE. Any of one or more alternative forms of a genetic sequence.Typically, in a diploid cell or organism, the two alleles of a givensequence typically occupy corresponding loci on a pair of homologouschromosomes.

BACKCROSSING. Process in which a breeder crosses a hybrid progeny lineback to one of the parental genotypes one or more times.

BREEDING. The genetic manipulation of living organisms.

BREEDING CROSS. A cross to introduce new genetic material into a plantfor the development of a new variety. For example, one could cross plantA with plant B, wherein plant B would be genetically different fromplant A. After the breeding cross, the resulting F1 plants could then beselfed or sibbed for one, two, three or more times (F1, F2, F3, etc.)until a new inbred variety is developed. For clarification, such newinbred varieties would be within a pedigree distance of one breedingcross of plants A and B. The process described above would be referredto as one breeding cycle.

CROSS POLLINATION. A plant is cross pollinated if the pollen comes froma flower on a different plant from a different family or line. Crosspollination excludes sib and self pollination.

CROSS. As used herein, the term “cross” or “crossing” can refer to asimple X by Y cross, or the process of backcrossing, depending on thecontext.

ELITE INBRED. An inbred that contributed desirable qualities when usedto produce commercial hybrids. An elite inbred may also be used infurther breeding for the purpose of developing further improvedvarieties.

INBRED. A line developed through inbreeding or doubled haploidy thatpreferably comprises homozygous alleles at about 95% or more of itsloci.

LINKAGE. Refers to a phenomenon wherein alleles on the same chromosometend to segregate together more often than expected by chance if theirtransmission was independent.

LINKAGE DISEQUILIBRIUM. Refers to a phenomenon wherein alleles tend toremain together in linkage groups when segregating from parents tooffspring, with a greater frequency than expected from their individualfrequencies.

LOCUS. A defined segment of DNA.

NEI DISTANCE. A quantitative measure of percent similarity between twolines. Nei's distance between lines A and B can be defined as1−(2*number alleles in common/(number alleles in A+number alleles in B).For example, if lines A and B are the same for 95 out of 100 alleles,the Nei distance would be 0.05. If lines A and B are the same for 98 outof 100 alleles, the Nei distance would be 0.02. Free software forcalculating Nei distance is available on the internet at multiplelocations such as, for example, at:evolution.genetics.washington.edu/phylip.html. See Nei, Proc Natl AcadSci, 76:5269-5273 (1979) which is incorporated by reference for thispurpose.

PEDIGREE DISTANCE. Relationship among generations based on theirancestral links as evidenced in pedigrees. May be measured by thedistance of the pedigree from a given starting point in the ancestry.

PERCENT IDENTITY. Percent identity as used herein refers to thecomparison of the homozygous alleles of two inbred lines. Each inbredplant will have the same allele (and therefore be homozygous) at almostall of their loci. Percent identity is determined by comparing astatistically significant number of the homozygous alleles of two inbredlines. For example, a percent identity of 90% between inbred PHWWE andother inbred line means that the two inbred lines have the same alleleat 90% of their loci.

PERCENT SIMILARITY. Percent similarity as used herein refers to thecomparison of the homozygous alleles of an inbred line with anotherplant. The homozygous alleles of PHWWE are compared with the alleles ofa non-inbred plant, such as a hybrid, and if the allele of the inbredmatches at least one of the alleles from the hybrid then they are scoredas similar. Percent similarity is determined by comparing astatistically significant number of loci and recording the number ofloci with similar alleles as a percentage. For example, a percentsimilarity of 90% between inbred PHWWE and a hybrid maize plant meansthat the inbred line matches at least one of the hybrid alleles at 90%of the loci. In the case of a hybrid produced from PHWWE as the male orfemale parent, such hybrid will comprise two sets of alleles, one set ofwhich will comprise the same alleles as the homozygous alleles of inbredline PHWWE.

PLANT. As used herein, the term “plant” includes reference to animmature or mature whole plant, including a plant that has beendetasseled or from which seed or grain has been removed. Seed or embryothat will produce the plant is also considered to be the plant.

SELF POLLINATION. A plant is self-pollinated if pollen from one floweris transferred to the same or another flower of the same plant.

SIB POLLINATION. A plant is sib-pollinated when individuals within thesame family or line are used for pollination.

SINGLE LOCUS CONVERSION TRAIT. A trait that can be introgressed into acorn line through introgression and/or transformation of a single locus.Examples of such single locus traits include mutant genes, transgenesand native traits finely mapped to a single locus. One or more singlelocus conversion traits may be introduced into a single corn line.

DETAILED DESCRIPTION OF THE INVENTION AND FURTHER EMBODIMENTSMorphological and Physiological Characteristics of PHWWE

Inbred maize line PHWWE, can be reproduced by planting seeds of theline, growing the resulting maize plants under self-pollinating orsib-pollinating conditions with adequate isolation, and harvesting theresulting seed using techniques familiar to the agricultural arts.

Development of PHWWE

The development of PHWWE was initiated by crossing a Hi-II maize linewith pollen from PH09B. PH09B is an elite maize line described in U.S.Pat. No. 5,859,354 and having seed deposited with the ATCC and havingthe Deposit Number: 203085. The F1 embryos produced from this cross werecultured on medium. Embryos producing good type II callus and fastcallus growing response were selected and plants were regenerated fromthe selected callus lines. The regenerated plants were grown andbackcrossed with PH09B to produce BC1 embryos. These BC1 immatureembryos were isolated and placed on culture medium for selection of thebest type II callus and fastest growing callus. Plants were regeneratedfrom the selected callus cultures. When the plants flowered they wereself pollinated forming BC1S1 seed. The BC1S1 seeds were planted in thefield. Plants were selected that were morphologically close to inbredPH09B. These selected plants were self-pollinated to produce BC1S2immature embryos. The BC1S2 through BC1S5 embryos were screened for thefollowing characters: 1) ability to receive T-DNA from Agrobacteriuminfection, 2) ability to initiate callus response followingAgrobacterium infection, 3) ability to produce fast-growing and friabletype II callus, 4) ability of callus tissue to be maintained on culturemedium for at least 3 months, 5) ability of callus tissues to regeneratefertile plants and 6) ability of regenerated plants to produce viableseed and good seed set. The immature embryos in these 4 generations wereinfected with Agrobacterium LBA4404 comprising a visible marker, such asCRC or GFP. After infection with the Agrobacterium the embryos werecultured on callus induction medium without selection pressure andevaluated at 7 days for transient expression of the visible marker.Since there was no selection pressure in the medium, both transformedand non-transformed cells could initiate callus response and the visiblemarker expressed only in the transformed cells and callus, but did notexpress in the non-transformed cells and callus. Embryos with the besttransient expression and best callus response were selected and theparts of the callus tissues that did not express the visible marker onthese selected embryos were used to regenerate into plants. Plantmorphology was evaluated for each generation. The plants morphologicallysimilar to PH09B were selected. The selected plants were self pollinatedto produce immature embryos and seeds for the next generation.

The immature embryos in the next three generations (BC1S6-BC1S8) weretested for stable transformation. The non-transformed seed from theparents that produced the embryos with the best callus quality,transformation efficiency and regeneration capability were selected andplanted; and the resulted plants were used for self-pollination togenerate the next generation. At this point, this line had beenself-pollinated for 8 generations and was therefore considered an inbredline.

Culture and Transformation Characteristics of PHWWE

Immature embryos isolated from PHWWE plants produce a high quality TypeII callus. Three criteria were used to measure the quality of the callustissue: callus initiation frequency, callus growth rate and plantregeneration efficiency. The callus initiation frequency of PHWWE isabout 80% or higher. The callus tissue produced from PHWWE embryos growsfaster than Hi-II (Table 1). The callus produced is very friable and ishighly embryogenic. In contrast, immature embryos from PH09B did notproduce Type II callus. PH09B plants produced embryos with low frequency(less than 3%) of compact Type I callus. This compact Type I callusgrows much slower than Type II callus.

TABLE 1 Comparison of Callus Growth Rate of PHWWE and Hi-II Line InitialWeight Weight at 4-Week Callus Growth Name Embryo (gm) Culture (gm) Rate(times) PHWWE 1 0.21 7.49 34.7 X 2 0.21 7.61 35.2 X 3 0.24 7.88 31.8 X 40.20 6.70 32.5 X 5 0.22 8.22 36.4 X 6 0.23 8.49 35.9 X 7 0.24 8.92 36.2X 8 0.21 8.43 39.1 X Average 0.22 7.97 35.2 X Hi-II 1 0.50 10.67 20.3 X2 0.44 11.77 25.8 X 3 0.48 13.05 26.2 X 4 0.40 10.40 25.0 X 5 0.32 9.9730.2 X Average 0.43 11.17 25.0 X

The data presented in Table 1 demonstrates the callus growth rate ofPHWWE and Hi-II. The initial cultures were weighed as were the 4-weekcultures on maintenance medium at 28 C in the dark the callus tissues.The callus weight of PHWWE increased 35.2 times of the callus weight ofinitial cultures while the callus weight of Hi-II increased 25 times.Callus growth rate was calculated as: Callus growth rate=(callus weightat 4-week−callus weight at initial culture)/callus weight at initialculture.

The immature embryos isolated from PHWWE plants were used forAgrobacterium-mediated transformation and microprojectile bombardmenttransformation. Two selection marker genes, bar and GAT, were used toselect stable transformants. The data listed in Table 2 showed theoverall transformation frequencies were 29% with bar selection(tolerance for glufosonate) and 28% with GAT selection (tolerance forglyphosate) when Agrobacterium-mediated transformation was used. Whenparticle bombardment was used with bar selection the rate of stabletransformation was 34.5%.

Embryos from PH09B and Hi-II×PH09B were used as controls. Thetransformation frequency of PH09B with Agrobacterium was zero percentand the transformation frequency of Hi-II×PH09B was less than 0.3%.

TABLE 2 Stable Transformation Experiments with PHWWE Total SelectionStable Callus Regeneration Overall Experiment Method Embryos MarkerEvents (%) (%) Frequency 1 Agro 304 Bar 91 (30%)   97% 29.1% 2 Agro 242Bar 74 (30.6%) 96% 29.4% 3 Agro 250 GAT 82 (32.8%) 85% 27.9% 4 Gun 2,386Bar 945 (39.6%)  87% 34.5%

Morphological Characteristics of PHWWE

PHWWE has been characterized agronomically both in the field and in thegreenhouse. When grown in the field at Johnston, Iowa it reached 50%pollen shed and 50% silk at 1550 Growing Degree Units (GDU) and 1620 GDUrespectively. PHWWE had an average plant height of 210 cm and averageear height of 82 cm. PHWWE has yellow dent kernels and purple cob andproduced about 256 seeds/ear on average. When it was grown in greenhouseconditions, PHWWE averaged 247 cm in height and produced about 308kernels/ear. In the greenhouse PHWWE averaged about 70 days fromplanting to flowering and about 105 days from planting to harvest.

Genotypic Characteristics of PHWWE

Molecular markers were used to analyze the genetic make-up of PHWWE. 289SSR markers (Table 3) that were polymorphic between PH09B and Hi-II wereused for the analysis. Using markers it was determined that the PHWWEgenome, derived about 36.6% of its gemone from Hi-II and about 63.4% ofits genome from PH09B.

A plant can be identified by its genotype. The genotype of a plant canbe characterized through a genetic marker profile, which can identifyplants of the same variety or a related variety, or be used to determineor validate a pedigree. The SSR profile of Inbred PHWWE can be found inTable 3.

As a result of the selfing process, PHWWE is substantially homozygous.This homozygosity has been characterized at the loci shown in the markerprofile provided herein. An F1 hybrid made with PHWWE would comprise themarker profile of PHWWE shown herein. This is because an F1 hybrid isthe sum of its inbred parents, e.g., if one inbred parent is homozygousfor allele x at a particular locus, and the other inbred parent ishomozygous for allele y at that locus, the F1 hybrid will be x.y(heterozygous) at that locus. The profile can therefore be used toidentify hybrids comprising PHWWE as a parent, since such hybrids willcomprise two sets of alleles, one set of which will be from PHWWE. Thedetermination of the male set of alleles and the female set of allelesmay be made by profiling the hybrid and the pericarp of the hybrid seed,which is composed of maternal parent cells. One way to obtain thepaternal parent profile is to subtract the pericarp profile from thehybrid profile.

Subsequent generations of progeny produced by selection and breeding areexpected to be of genotype x (homozygous), y (homozygous), or x.y(heterozygous) for these locus positions. When the F1 plant is used toproduce an inbred, the resulting inbred should be either x or y for thatallele. In that regard, a unique allele or combination of alleles uniqueto that inbred can be used to identify progeny plants that retain thoseunique alleles or combinations of alleles.

Therefore, in accordance with the above, an embodiment of this inventionis a PHWWE progeny maize plant or plant part that is a first generation(F1) hybrid maize plant comprising two sets of alleles, wherein one setof the alleles is the same as PHWWE at all of the SSR loci listed inTable 3. A maize cell wherein one set of the alleles is the same asPHWWE at all of the SSR loci listed in Table 3 is also an embodiment ofthe invention. This maize cell may be a part of a hybrid seed, plant orplant part produced by crossing PHWWE with another inbred maize plant.

Genetic marker profiles can be obtained by techniques such asRestriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), Simple Sequence Repeats (SSRs) which are also referred to asMicrosatellites, and Single Nucleotide Polymorphisms (SNPs). Forexample, see Berry, Don et al., “Assessing Probability of Ancestry UsingSimple Sequence Repeat Profiles: Applications to Maize Hybrids andInbreds”, Genetics, 2002, 161:813-824, and Berry, Don et al., “AssessingProbability of Ancestry Using Simple Sequence Repeat Profiles:Applications to Maize Inbred Lines and Soybean Varieties”, Genetics,2003, 165:331-342.

Particular markers used for these purposes are not limited to the set ofmarkers disclosed herein, but may include any type of marker and markerprofile which provides a means of distinguishing varieties. In additionto being used for identification of maize line PHWWE, a hybrid producedthrough the use of PHWWE, and the identification or verification ofpedigree for progeny plants produced through the use of PHWWE, thegenetic marker profile is also useful in further breeding and indeveloping an introgressed trait conversion of PHWWE.

Means of performing genetic marker profiles using SSR polymorphisms arewell known in the art. SSRs are genetic markers based on polymorphismsin repeated nucleotide sequences, such as microsatellites. A markersystem based on SSRs can be highly informative in linkage analysisrelative to other marker systems in that multiple alleles may bepresent. Another advantage of this type of marker is that, through useof flanking primers, detection of SSRs can be achieved, for example, bythe polymerase chain reaction (PCR), thereby eliminating the need forlabor-intensive Southern hybridization. The PCR detection is done by useof two oligonucleotide primers flanking the polymorphic segment ofrepetitive DNA. Repeated cycles of heat denaturation of the DNA followedby annealing of the primers to their complementary sequences at lowtemperatures, and extension of the annealed primers with DNA polymerase,comprise the major part of the methodology.

Following amplification, markers can be scored by electrophoresis of theamplification products. Scoring of marker genotype is based on the sizeof the amplified fragment, which may be measured by the number of basepairs of the fragment. While variation in the primer used or inlaboratory procedures can affect the reported fragment size, relativevalues should remain constant regardless of the specific primer orlaboratory used. When comparing lines it is preferable if all SSRprofiles are performed in the same lab. The SSR analyses reported hereinwere conducted in-house at Pioneer Hi-Bred. An SSR service is availableto the public on a contractual basis by DNA Landmarks inSaint-Jean-sur-Richelieu, Quebec, Canada.

Primers used for the SSRs reported herein are publicly available and maybe found in the Maize GDB on the World Wide Web at maizegdb.org(sponsored by the USDA Agricultural Research Service), in Sharopova etal. (Plant Mol. Biol. 48(5-6):463-481), Lee et al. (Plant Mol. Biol.48(5-6); 453-461), or may be constructed from sequences if reportedherein. Primers may be constructed from publicly available sequenceinformation. Some marker information may also be available from DNALandmarks.

Map information is provided by bin number as reported in the Maize GDBfor the IBM 2 and/or IBM 2 Neighbors maps. The bin number digits to theleft of decimal point represent the chromosome on which such marker islocated, and the digits to the right of the decimal represent thelocation on such chromosome. Map positions are also available on theMaize GDB for a variety of different mapping populations.

PHWWE and its plant parts can be identified through a molecular markerprofile. An inbred corn plant cell having the SSR genetic marker profileshown in Table 3 is an embodiment of the invention. Such plant cell maybe either diploid or haploid.

Also encompassed within the scope of the invention are plants and plantparts substantially benefiting from the use of PHWWE in theirdevelopment, such as PHWWE comprising a introgressed trait throughbackcross conversion or transformation, and which may be identified byhaving an SSR molecular marker profile with a high percent identity toPHWWE, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity.Likewise, percent similarity at these percentages may be used toidentify hybrid and other non-inbred plants produced by the use ofPHWWE.

An embodiment of this invention is an inbred PHWWE progeny maize plantor plant part comprising the same homozygous alleles as the plant orplant part of PHWWE for at least 90% of the SSR loci listed in Table 3.A plant cell comprising the same homozygous alleles as a plant cell ofPHWWE for at least 90% of the SSR loci listed in Table 3 is also anembodiment of this invention. In these specific embodiments, 90% mayalso be replaced by any integer or partial integer percent of 80% orgreater as listed above. One means of producing such a progeny plant,plant part or cell is through the backcrossing and/or transformationmethods described herein.

Similarly, an embodiment of this invention is a PHWWE progeny maizeplant or plant part comprising at least one allele per locus that is thesame allele as the plant or plant part of PHWWE for at least 90% of theSSR loci listed in Table 3. This progeny plant may be a hybrid. Aprogeny or hybrid plant cell wherein at least one allele per locus thatis the same allele as the plant cell PHWWE for at least 90% of the SSRloci listed in Table 3 is also a specific embodiment of this invention.In these specific embodiments, 90% may also be replaced by any integerpercent listed above. One means of producing such a progeny or hybridplant, plant part or cell is through the backcrossing and/ortransformation methods described herein.

In addition, the SSR profile of PHWWE also can be used to identifyessentially derived varieties and other progeny lines developed from theuse of PHWWE, as well as cells and other plant parts thereof. Progenyplants and plant parts produced using PHWWE may be identified by havinga molecular marker profile of at least 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%genetic contribution from inbred line PHWWE, as measured by eitherpercent identity or percent similarity.

Comparing PHWWE to Other Inbreds

A breeder uses various methods to help determine which plants should beselected from segregating populations and ultimately which inbred lineswill be used to develop hybrids for commercialization. In addition toknowledge of the germplasm and plant genetics, a part of the selectionprocess is dependent on experimental design coupled with the use ofstatistical analysis. Experimental design and statistical analysis areused to help determine which plants, which family of plants, and finallywhich inbred lines and hybrid combinations are significantly better ordifferent for one or more traits of interest. Experimental designmethods are used to assess error so that differences between two inbredlines or two hybrid lines can be more accurately evaluated. Statisticalanalysis includes the calculation of mean values, determination of thestatistical significance of the sources of variation, and thecalculation of the appropriate variance components. Either a five or aone percent significance level is customarily used to determine whethera difference that occurs for a given trait is real or due to theenvironment or experimental error. One of ordinary skill in the art ofplant breeding would know how to evaluate the traits of two plantvarieties to determine if there is no significant difference between thetwo traits expressed by those varieties. For example, see Fehr, Walt,Principles of Cultivar Development, p. 261-286 (1987). Mean trait valuesmay be used to determine whether trait differences are significant.Trait values should preferably be measured on plants grown under thesame environmental conditions, and environmental conditions should beappropriate for the traits or traits being evaluated.

Development of Maize Hybrids using PHWWE

A single cross maize hybrid results from the cross of two inbred lines,each of which has a genotype that complements the genotype of the other.The hybrid progeny of the first generation is designated F1. PHWWE maybe used to produce hybrid maize. One such embodiment is the method ofcrossing inbred maize line PHWWE with another maize plant, such as adifferent maize inbred line, to form a first generation F1 hybrid seed.The first generation F1 hybrid seed, plant and plant part produced bythis method is an embodiment of the invention. The first generation F1seed, plant and plant part will comprise an essentially complete set ofthe alleles of inbred line PHWWE. One of ordinary skill in the art canutilize either breeder books or molecular methods to identify aparticular F1 hybrid plant produced using inbred line PHWWE. Further,one of ordinary skill in the art may also produce F1 hybrids withtransgenic, male sterile and/or backcross conversions of inbred linePHWWE.

PHWWE may be used to produce a single cross hybrid, a double crosshybrid, or a three-way hybrid. A single cross hybrid is produced whentwo inbred lines are crossed to produce the F1 progeny. A double crosshybrid is produced from four inbred lines crossed in pairs (A×B and C×D)and then the two F1 hybrids are crossed again (A×B)×(C×D). A three-waycross hybrid is produced from three inbred lines where two of the inbredlines are crossed (A×B) and then the resulting F1 hybrid is crossed withthe third inbred (A×B)×C. In each case, pericarp tissue from the femaleparent will be a part of and protect the hybrid seed.

Male Sterility and Hybrid Seed Production

Hybrid seed production requires elimination or inactivation of pollenproduced by the female inbred parent. Incomplete removal or inactivationof the pollen provides the potential for self-pollination. A reliablemethod of controlling male fertility in plants offers the opportunityfor improved seed production.

PHWWE can be produced in a male-sterile form. There are several ways inwhich a maize plant can be manipulated so that it is male sterile. Theseinclude use of manual or mechanical emasculation (or detasseling), useof one or more genetic factors that confer male sterility, includingcytoplasmic genetic and/or nuclear genetic male sterility, use ofgametocides and the like. A male sterile inbred designated PHWWE mayinclude one or more genetic factors, which result in cytoplasmic geneticand/or nuclear genetic male sterility. All of such embodiments arewithin the scope of the present claims. The male sterility may be eitherpartial or complete male sterility.

Hybrid maize seed is often produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twomaize inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Provided that there issufficient isolation from sources of foreign maize pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

The laborious detasseling process can be avoided by using cytoplasmicmale-sterile (CMS) inbreds. Plants of a CMS inbred are male sterile as aresult of genetic factors in the cytoplasm, as opposed to the nucleus,and so nuclear linked genes are not transferred during backcrossing.Thus, this characteristic is inherited exclusively through the femaleparent in maize plants, since only the female provides cytoplasm to thefertilized seed. CMS plants are fertilized with pollen from anotherinbred that is not male-sterile. Pollen from the second inbred may ormay not contribute genes that make the hybrid plants male-fertile, andeither option may be preferred depending on the intended use of thehybrid. The same hybrid seed, a portion produced from detasseled fertilemaize and a portion produced using the CMS system, can be blended toinsure that adequate pollen loads are available for fertilization whenthe hybrid plants are grown. CMS systems have been successfully usedsince the 1950's, and the male sterility trait is routinely backcrossedinto inbred lines. See Wych, “Production of Hybrid Seed”, Corn and CornImprovement, Ch. 9, pp. 565-607, 1998.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

These, and the other methods of conferring genetic male sterility in theart, each possess their own benefits and drawbacks. Some other methodsuse a variety of approaches such as delivering into the plant a geneencoding a cytotoxic substance associated with a male tissue specificpromoter or an antisense system in which a gene critical to fertility isidentified and an antisense to that gene is inserted in the plant (seeFabinjanski, et al. EPO 89/3010153.8 publication no. 329,308 and PCTapplication PCT/CA90/00037 published as WO 90/08828).

Another system for controlling male sterility makes use of gametocides.Gametocides are not a genetic system, but rather a topical applicationof chemicals. These chemicals affect cells that are critical to malefertility. The application of these chemicals affects fertility in theplants only for the growing season in which the gametocide is applied(see Carlson, Glenn R., U.S. Pat. No. 4,936,904). Application of thegametocide, timing of the application and genotype specificity oftenlimit the usefulness of the approach and it is not appropriate in allsituations.

Introgression of a New Locus or Trait into PHWWE

PHWWE represents a new base genetic line into which a new locus or traitmay be introgressed. Direct transformation and backcrossing representtwo important methods that can be used to accomplish such anintrogression. The term backcross conversion and single locus conversionare used interchangeably to designate the product of a backcrossingprogram.

Backcross Conversions of PHWWE

A backcross conversion of PHWWE occurs when DNA sequences are introducedthrough backcrossing (Hallauer et al. in Corn and Corn Improvement,Sprague and Dudley, Third Ed. 1998), with PHWWE utilized as therecurrent parent. Both naturally occurring and transgenic DNA sequencesmay be introduced through backcrossing techniques. A backcrossconversion may produce a plant with a trait or locus conversion in atleast one or more backcrosses, including at least 2 crosses, at least 3crosses, at least 4 crosses, at least 5 crosses and the like. Molecularmarker assisted breeding or selection may be utilized to reduce thenumber of backcrosses necessary to achieve the backcross conversion. Forexample, see Openshaw, S. J. et al., Marker-assisted Selection inBackcross Breeding, In: Proceedings Symposium of the Analysis ofMolecular Data, August 1994, Crop Science Society of America, Corvallis,Oreg., where it is demonstrated that a backcross conversion can be madein as few as two backcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes as vs.unlinked genes), the level of expression of the trait, the type ofinheritance (cytoplasmic or nuclear) and the types of parents includedin the cross. It is understood by those of ordinary skill in the artthat for single gene traits that are relatively easy to classify, thebackcross method is effective and relatively easy to manage. (SeeHallauer et al. in Corn and Corn Improvement, Sprague and Dudley, ThirdEd. 1998). Desired traits that may be transferred through backcrossconversion include, but are not limited to, waxy starch, sterility(nuclear and cytoplasmic), fertility restoration, grain color (white),nutritional enhancements, drought resistance, enhanced nitrogenutilization efficiency, altered nitrogen responsiveness, altered fattyacid profile, increased digestibility, low phytate, industrialenhancements, disease resistance (bacterial, fungal or viral), insectresistance, herbicide resistance and yield enhancements. In addition, anintrogression site itself, such as an FRT site, Lox site or other sitespecific integration site, may be inserted by backcrossing and utilizedfor direct insertion of one or more genes of interest into a specificplant variety. In some embodiments of the invention, the number of locithat may be backcrossed into PHWWE is at least 1, 2, 3, 4, or 5 and/orno more than 6, 5, 4, 3, or 2. A single loci may contain severaltransgenes, such as a transgene for disease resistance that, in the sameexpression vector, also contains a transgene for herbicide resistance.The gene for herbicide resistance may be used as a selectable markerand/or as a phenotypic trait. A single locus conversion of site specificintegration (SSI) system allows for the integration of multiple genes atthe converted loci. Further, SSI technologies known to those of skill inthe art in the art may result in multiple gene introgressions at asingle locus.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele, such as the waxy starchcharacteristic, requires growing and selfing the first backcrossgeneration to determine which plants carry the recessive alleles.Recessive traits may require additional progeny testing in successivebackcross generations to determine the presence of the locus ofinterest. The last backcross generation is usually selfed to give purebreeding progeny for the gene(s) being transferred, although a backcrossconversion with a stably introgressed trait may also be maintained byfurther backcrossing to the recurrent parent with selection for theconverted trait.

Along with selection for the trait of interest, progeny are selected forthe phenotype of the recurrent parent. While occasionally additionalpolynucleotide sequences or genes may be transferred along with thebackcross conversion, the backcross conversion line “fits into the samehybrid combination as the recurrent parent inbred line and contributesthe effect of the additional gene added through the backcross.” Poehlmanet al. (1995) Breeding Field Crop, 4th Ed., Iowa State University Press,Ames, Iowa., pp. 132-155 and 321-344. It has been proposed that ingeneral there should be at least four backcrosses when it is importantthat the recovered lines be essentially identical to the recurrentparent except for the characteristic being transferred (Fehr 1987,Principles of Cultivar Development). However, as noted above, the numberof backcrosses necessary can be reduced with the use of molecularmarkers. Other factors, such as a genetically similar donor parent, mayalso reduce the number of backcrosses necessary.

One process for adding or modifying a trait or locus in maize inbredline PHWWE comprises crossing PHWWE plants grown from PHWWE seed withplants of another maize line that comprise the desired trait or locus,selecting F1 progeny plants that comprise the desired trait or locus toproduce selected F1 progeny plants, crossing the selected progeny plantswith the PHWWE plants to produce backcross progeny plants, selecting forbackcross progeny plants that have the desired trait or locus and themorphological characteristics of maize inbred line PHWWE to produceselected backcross progeny plants; and backcrossing to PHWWE three ormore times in succession to produce selected fourth or higher backcrossprogeny plants that comprise said trait or locus. The modified PHWWE maybe further characterized as having the physiological and morphologicalcharacteristics of maize inbred line PHWWE. Differences in physiologicaland morphological characteristics can be determined at the 5%significance level when grown in the same environmental conditionsand/or may be characterized by percent similarity or identity to PHWWEas determined by SSR markers. The above method may be utilized withfewer backcrosses in appropriate situations, such as when the donorparent is highly related or markers are used in the selection step.Desired traits that may be used include those nucleic acids known in theart, some of which are listed herein, that will affect traits throughnucleic acid expression or inhibition. Desired loci include theintrogression of FRT, Lox and other sites for site specific integration.

In addition, the above process and other similar processes describedherein may be used to produce F1 hybrid maize seed by adding a step atthe end of the process that comprises crossing PHWWE with theintrogressed trait or locus with a different maize plant and harvestingthe resultant F1 hybrid maize seed.

Introgression of a New Locus or Trait into PHWWE through Transformation

Transformation of a PHWWE cell may also be used in the methods. The typeof transformation is not critical to the methods; various methods oftransformation are currently available. As newer methods are availableto transform host cells they may be directly applied. Accordingly, awide variety of methods have been developed to insert a DNA sequenceinto the genome of a host cell to obtain the transcription and/ortranslation of the sequence. Thus, any method that provides forefficient transformation/transfection may be employed.

Methods for transforming various host cells are disclosed in Klein etal. “Transformation of microbes, plants and animals by particlebombardment”, Bio/Technol. New York, N.Y., Nature Publishing Company,March 1992, 10(3):286-291. Techniques for transforming a wide variety ofhigher plant species are well known and described in the technical,scientific, and patent literature. See, for example, Weising et al.,Ann. Rev. Genet. 22:421-477 (1988).

For example, the DNA construct may be introduced directly into thegenomic DNA of the plant cell using techniques such as electroporation,PEG-induced transfection, particle bombardment, silicon fiber delivery,or microinjection of plant cell protoplasts or embryogenic callus. See,e.g., Tomes et al., Direct DNA Transfer into Intact Plant Cells ViaMicroprojectile Bombardment. pp. 197-213 in Plant Cell, Tissue and OrganCulture, Fundamental Methods. eds. O. L. Gamborg and G. C. Phillips.Springer-Verlag Berlin Heidelberg New York, 1995. The introduction ofDNA constructs using polyethylene glycol precipitation is described inPaszkowski et al., Embo J. 3:2717-2722 (1984). Electroporationtechniques are described in Fromm et al., Proc. Natl. Acad. Sci. 82:5824(1985). Ballistic transformation techniques are described in Klein etal., Nature 327:70-73 (1987). An aerosol transformation method isdisclosed in U.S. Pat. No. 7,001,754.

Alternatively, the DNA constructs may be combined with suitable T-DNAflanking regions and introduced into a Agrobacterium tumefaciens hostvector. The virulence functions of the Agrobacterium tumefaciens hostwill direct the insertion of the construct and adjacent marker into theplant cell DNA when the cell is infected by the bacteria. Agrobacteriumtumefaciens-meditated transformation techniques are well described inthe scientific literature. See, for example Horsch et al., Science233:496-498 (1984), and Fraley et al., Proc. Natl. Acad. Sci. 80:4803(1983). For instance, Agrobacterium transformation of maize is describedin U.S. Pat. No. 5,981,840. Agrobacterium transformation of monocot isfound in U.S. Pat. No. 5,591,616. Agrobacterium transformation ofsoybeans is described in U.S. Pat. No. 5,563,055.

Other methods of transformation include (1) Agrobacteriumrhizogenes-induced transformation (see, e.g., Lichtenstein and FullerIn: Genetic Engineering, vol. 6, P W J Rigby, Ed., London, AcademicPress, 1987; and Lichtenstein, C. P., and Draper, J,. In: DNA Cloning,Vol. II, D. M. Glover, Ed., Oxford, IRI Press, 1985), ApplicationPCT/US87/02512 (WO 88/02405 published Apr. 7, 1988) describes the use ofA. rhizogenes strain A4 and its Ri plasmid along with A. tumefaciensvectors pARC8 or pARC16 (2) liposome-induced DNA uptake (see, e.g.,Freeman et al., Plant Cell Physiol. 25:1353, 1984), (3) the vortexingmethod (see, e.g., Kindle, Proc. Natl. Acad. Sci., USA 87:1228, (1990).

DNA can also be introduced into plants by direct DNA transfer intopollen as described by Zhou et al., Methods in Enzymology 101:433(1983); D. Hess, Intern Rev. Cytol. 107:367 (1987); Luo et al., PlantMol. Biol. Reporter, 6:165 (1988). Expression of polypeptide codingnucleic acids can be obtained by injection of the DNA into reproductiveorgans of a plant as described by Pena et al., Nature 325:274 (1987).Transformation can also be achieved through electroporation of foreignDNA into sperm cells then microinjecting the transformed sperm cellsinto isolated embryo sacs as described in U.S. Pat. No. 6,300,543 byCass et al. DNA can also be injected directly into the cells of immatureembryos and the rehydration of desiccated embryos as described byNeuhaus et al., Theor. Appl. Genet. 75:30 (1987); and Benbrook et al.,in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54(1986).

Transformed cells which are derived by any of the above transformationtechniques can be cultured to regenerate a whole plant which possessesthe transformed genotype. For transformation and regeneration of maizesee, Gordon-Kamm et al., The Plant Cell 2:603-618 (1990).

Any DNA sequences, whether from a different species or from the samespecies, which are inserted into the genome using transformation arereferred to herein collectively as “transgenes”. In some embodiments ofthe invention, a transformed variant of PHWWE may contain at least onetransgene but could contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.Over the last fifteen to twenty years several methods for producingtransgenic plants have been developed, and the present invention alsorelates to transformed versions of the claimed inbred maize line PHWWEas well as hybrid combinations thereof.

The most prevalent types of plant transformation involve theconstruction of an expression vector. Such a vector comprises a DNAsequence that contains a gene under the control of or operatively linkedto a regulatory element, for example a promoter. The vector may containone or more genes and one or more regulatory elements.

A genetic trait which has been engineered into the genome of aparticular maize plant using transformation techniques, could be movedinto the genome of another line using traditional breeding techniquesthat are well known in the plant breeding arts. For example, abackcrossing approach is commonly used to move a transgene from atransformed maize plant to an elite inbred line, and the resultingprogeny would then comprise the transgene(s). Also, if an inbred linewas used for the transformation then the transgenic plants could becrossed to a different inbred in order to produce a transgenic hybridmaize plant.

Various genetic elements can be introduced into the plant genome usingtransformation. These elements include, but are not limited to genes;coding sequences; inducible, constitutive, and tissue specificpromoters; enhancing sequences; and signal and targeting sequences. Forexample, see the traits, genes and transformation methods listed in U.S.Pat. Nos. 6,118,055 and 6,284,953, which are herein incorporated byreference.

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants that areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114: 92-6(1981).

A genetic map can be generated, primarily via conventional RestrictionFragment Length Polymorphisms (RFLP), Polymerase Chain Reaction (PCR)analysis, Simple Sequence Repeats (SSR) and Single NucleotidePolymorphisms (SNP) that identifies the approximate chromosomal locationof the integrated DNA molecule. For exemplary methodologies in thisregard, see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology 269-284 (CRC Press, Boca Raton, 1993).

Wang et al. discuss “Large Scale Identification, Mapping and Genotypingof Single-Nucleotide Polymorphisms in the Human Genome”, Science,280:1077-1082, 1998, and similar capabilities are available for the corngenome. Map information concerning chromosomal location is useful forproprietary protection of a subject transgenic plant. If unauthorizedpropagation is undertaken and crosses made with other germplasm, the mapof the integration region can be compared to similar maps for suspectplants to determine if the latter have a common parentage with thesubject plant. Map comparisons would involve hybridizations, RFLP, PCR,SSR and sequencing, all of which are conventional techniques. SNPs mayalso be used alone or in combination with other techniques.

Likewise, by means of the present invention, plants can be geneticallyengineered to express various phenotypes of agronomic interest. Throughthe transformation of maize the expression of genes can be altered toenhance disease resistance, insect resistance, herbicide resistance,agronomic traits, grain quality and other traits. Transformation canalso be used to insert DNA sequences which control or help controlmale-sterility. DNA sequences native to maize as well as non-native DNAsequences can be transformed into maize and used to alter levels ofnative or non-native proteins. Various promoters, targeting sequences,enhancing sequences, and other DNA sequences can be inserted into themaize genome for the purpose of altering the expression of proteins.Reduction of the activity of specific genes (also known as genesilencing, or gene suppression) is desirable for several aspects ofgenetic engineering in plants.

Many techniques for gene silencing are well known to one of skill in theart, including but not limited to knock-outs (such as by insertion of atransposable element such as mu (Vicki Chandler, The Maize Handbook ch.118 (Springer-Verlag 1994) or other genetic elements such as a FRT, Loxor other site specific integration site, antisense technology (see,e.g., Sheehy et al. (1988) PNAS USA 85:8805-8809; and U.S. Pat. Nos.5,107,065; 5,453, 566; and 5,759,829); co-suppression (e.g., Taylor(1997) Plant Cell 9:1245; Jorgensen (1990) Trends Biotech.8(12):340-344; Flavell (1994) PNAS USA 91:3490-3496; Finnegan et al.(1994) Bio/Technology 12: 883-888; and Neuhuber et al. (1994) Mol. Gen.Genet. 244:230-241); RNA interference (Napoli et al. (1990) Plant Cell2:279-289; U.S. Pat. No. 5,034,323; Sharp (1999) Genes Dev. 13:139-141;Zamore et al. (2000) Cell 101:25-33; and Montgomery et al. (1998) PNASUSA 95:15502-15507), virus-induced gene silencing (Burton, et al. (2000)Plant Cell 12:691-705; and Baulcombe (1999) Curr. Op. Plant Bio.2:109-113); target-RNA-specific ribozymes (Haseloff et al. (1988) Nature334: 585-591); hairpin structures (Smith et al. (2000) Nature407:319-320; WO 99/53050; and WO 98/53083); MicroRNA (Aukerman & Sakai(2003) Plant Cell 15:2730-2741); ribozymes (Steinecke et al. (1992) EMBOJ. 11:1525; and Perriman et al. (1993) Antisense Res. Dev. 3:253);oligonucleotide mediated targeted modification (e.g., WO 03/076574 andWO 99/25853); Zn-finger targeted molecules (e.g., WO 01/52620; WO03/048345; and WO 00/42219); and other methods or combinations of theabove methods known to those of skill in the art.

Exemplary nucleotide sequences that may be altered by geneticengineering include, but are not limited to, those categorized below.

1. Transgenes That Confer Resistance to Insects or Disease and ThatEncode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266: 789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae); McDowell & Woffenden, (2003) Trends Biotechnol.21(4): 178-83 and Toyoda et al., (2002) Transgenic Res. 11(6):567-82. Aplant resistant to a disease is one that is more resistant to a pathogenas compared to the wild type plant.

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence ofa Bt delta-endotoxin gene. Moreover, DNA molecules encodingdelta-endotoxin genes can be purchased from American Type CultureCollection (Rockville, Md.), for example, under ATCC Accession Nos.40098, 67136, 31995 and 31998. Other examples of Bacillus thuringiensistransgenes being genetically engineered are given in the followingpatents and patent applications and hereby are incorporated by referencefor this purpose: U.S. Pat. Nos. 5,188,960; 5,689,052; 5,880,275; WO91/14778; WO 99/31248; WO 01/12731; WO 99/24581; WO 97/40162 and U.S.application Ser. Nos. 10/032,717; 10/414,637; and 10/606,320.

(C) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344: 458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

(D) An insect-specific peptide which, upon expression, disrupts thephysiology of the affected pest. For example, see the disclosures ofRegan, J. Biol. Chem. 269: 9 (1994) (expression cloning yields DNAcoding for insect diuretic hormone receptor); Pratt et al., Biochem.Biophys. Res. Comm. 163: 1243 (1989) (an allostatin is identified inDiploptera puntata); Chattopadhyay et al. (2004) Critical Reviews inMicrobiology 30 (1): 33-54 2004; Zjawiony (2004) J Nat Prod 67 (2):300-310; Carlini & Grossi-de-Sa (2002) Toxicon, 40 (11): 1515-1539;Ussuf et al. (2001) Curr Sci. 80 (7): 847-853; and Vasconcelos &Oliveira (2004) Toxicon 44 (4): 385-403. See also U.S. Pat. No.5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific toxins.

(E) An enzyme responsible for a hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(F) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21: 673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene, U.S. application Ser. Nos.10/389,432, 10/692,367, and U.S. Pat. No. 6,563,020.

(G) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104: 1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

(H) A hydrophobic moment peptide. See PCT application WO 95/16776 andU.S. Pat. No. 5,580,852 (disclosure of peptide derivatives ofTachyplesin which inhibit fungal plant pathogens) and PCT application WO95/18855 and U.S. Pat. No. 5,607,914) (teaches synthetic antimicrobialpeptides that confer disease resistance).

(I) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993),of heterologous expression of a cecropin-beta lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(J) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28: 451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus.

(K) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULARPLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

(L) A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366: 469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(M) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo alpha-1,4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-alpha-1,4-D-galacturonase. See Lamb etal., Bio/Technology 10: 1436 (1992). The cloning and characterization ofa gene which encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2: 367 (1992).

(N) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10: 305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, S., Current Biology,5(2):128-131 (1995), Pieterse & Van Loon (2004) Curr. Opin. Plant Bio.7(4):456-64 and Somssich (2003) Cell 113(7):815-6.

(P) Antifungal genes (Cornelissen and Melchers, PI. Physiol.101:709-712, (1993) and Parijs et al., Planta 183:258-264, (1991) andBushnell et al., Can. J. of Plant Path. 20(2):137-149 (1998). Also seeU.S. application Ser. No.: 09/950,933.

(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see U.S. Pat. No. 5,792,931.

(R) Cystatin and cysteine proteinase inhibitors. See U.S. applicationSer. No.: 10/947,979.

(S) Defensin genes. See WO03000863 and U.S. application Ser. No.:10/178,213.

(T) Genes conferring resistance to nematodes. See WO 03/033651 and Urwinet. al., Planta 204:472-479 (1998), Williamson (1999) Curr Opin PlantBio. 2(4):327-31.

(U) Genes such as rcg1 conferring resistance to Anthracnose stalk rot,which is caused by the fungus Colletotrichum graminiola. See M. Jung etal., Generation-means analysis and quantitative trait locus mapping ofAnthracnose Stalk Rot genes in Maize, Theor. Appl. Genet. (1994)89:413-418 which is incorporated by reference for this purpose, as wellas U.S. Patent Application 60/675,664, which is also incorporated byreference for this purpose.

2. Transgenes That Confer Resistance to a Herbicide, For Example:

(A) A herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7: 1241 (1988), and Miki et al., Theor. Appl. Genet. 80: 449(1990), respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659;5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107;5,928,937; and 5,378,824; and international publication WO 96/33270,which are incorporated herein by reference for this purpose.

(B) Glyphosate (resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin acetyl transferase (bar) genes), andpyridinoxy or phenoxy proprionic acids and cycloshexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. U.S. Pat. No. 5,627,061 to Barryet al. also describes genes encoding EPSPS enzymes. See also U.S. Pat.Nos. 6,566,587; 6,338,961; 6,248,876 B1; 6,040,497; 5,804,425;5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835;5,866,775; 6,225,114 B1; 6,130,366; 5,310,667; 4,535,060; 4,769,061;5,633,448; 5,510,471; Re. 36,449; RE 37,287 E; and 5,491,288; andinternational publications EP1173580; WO 01/66704; EP1173581 andEP1173582, which are incorporated herein by reference for this purpose.Glyphosate resistance is also imparted to plants that express a genethat encodes a glyphosate oxido-reductase enzyme as described more fullyin U.S. Pat. Nos. 5,776,760 and 5,463,175, which are incorporated hereinby reference for this purpose. In addition glyphosate resistance can beimparted to plants by the over expression of genes encoding glyphosateN-acetyltransferase. See, for example, U.S. application Ser. Nos.US01/46,227; 10/427,692 and 10/427,692. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European Patent Application No. 0 333 033 to Kumadaet al. and U.S. Pat. No. 4,975,374 to Goodman et al. disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in European PatentNo. 0 242 246 and 0 242 236 to Leemans et al. De Greef et al.,Bio/Technology 7: 61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. See also, U.S. Pat. Nos. 5,969,213;5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477;5,646,024; 6,177,616 B1; and 5,879,903, which are incorporated herein byreference for this purpose. Exemplary genes conferring resistance tophenoxy proprionic acids and cycloshexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83: 435 (1992).

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

(D) Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants (see, e.g., Hattori et al. (1995)Mol Gen Genet 246:419). Other genes that confer resistance to herbicidesinclude: a gene encoding a chimeric protein of rat cytochrome P4507A1and yeast NADPH-cytochrome P450 oxidoreductase (Shiota et al. (1994)Plant Physiol. 106(1):17-23), genes for glutathione reductase andsuperoxide dismutase (Aono et al. (1995) Plant Cell Physiol 36:1687, andgenes for various phosphotransferases (Datta et al. (1992) Plant MolBiol 20:619).

(E) Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1;and 5,767,373; and international publication WO 01/12825.

3. Transgenes That Confer or Contribute to an Altered GrainCharacteristic, Such As:

(A) Altered fatty acids, for example, by

-   -   (1) Down-regulation of stearoyl-ACP desaturase to increase        stearic acid content of the plant. See Knultzon et al., Proc.        Natl. Acad. Sci. USA 89: 2624 (1992) and WO99/64579 (Genes for        Desaturases to Alter Lipid Profiles in Corn),    -   (2) Elevating oleic acid via FAD-2 gene modification and/or        decreasing linolenic acid via FAD-3 gene modification (see U.S.        Pat. Nos. 6,063,947; 6,323,392; 6,372,965 and WO 93/11245),    -   (3) Altering conjugated linolenic or linoleic acid content, such        as in WO 01/12800,    -   (4) Altering LEC1, AGP, Dek1, Superal1, mi1ps, various Ipa genes        such as Ipa1, Ipa3, hpt or hggt. For example, see WO 02/42424,        WO 98/22604, WO 03/011015, U.S. Pat. No. 6,423,886, U.S. Pat.        No. 6,197,561, U.S. Pat. No. 6,825,397, US2003/0079247,        US2003/0204870, WO02/057439, WO03/011015 and Rivera-Madrid, R.        et. al. Proc. Natl. Acad. Sci. 92:5620-5624 (1995).

(B) Altered phosphorus content, for example, by the

-   -   (1) Introduction of a phytase-encoding gene would enhance        breakdown of phytate, adding more free phosphate to the        transformed plant. For example, see Van Hartingsveldt et al.,        Gene 127: 87 (1993), for a disclosure of the nucleotide sequence        of an Aspergillus niger phytase gene.    -   (2) Up-regulation of a gene that reduces phytate content. In        maize, this, for example, could be accomplished, by cloning and        then re-introducing DNA associated with one or more of the        alleles, such as the LPA alleles, identified in maize mutants        characterized by low levels of phytic acid, such as in Raboy et        al., Maydica 35: 383 (1990) and/or by altering inositol kinase        activity as in WO 02/059324, US2003/0009011, WO 03/027243,        US2003/0079247, WO 99/05298, U.S. Pat. No. 6,197,561, U.S. Pat.        No. 6,291,224, U.S. Pat. No. 6,391,348, WO2002/059324,        US2003/0079247, WO98/45448, WO99/55882, WO01/04147.

(C) Altered carbohydrates effected, for example, by altering a gene foran enzyme that affects the branching pattern of starch or a genealtering thioredoxin such as NTR and/or TRX (see U.S. Pat. No. 6,531,648which is incorporated by reference for this purpose) and/or a gamma zeinknock out or mutant such as cs27 or TUSC27 or en27 (See U.S. Pat. No.6,858,778 and US2005/0160488, US2005/0204418; which are incorporated byreference for this purpose). See Shiroza et al., J. Bacteriol. 170: 810(1988) (nucleotide sequence of Streptococcus mutans fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 200: 220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al.,Bio/Technology 10: 292 (1992) (production of transgenic plants thatexpress Bacillus licheniformis alpha-amylase), Elliot et al., PlantMolec. Biol. 21: 515 (1993) (nucleotide sequences of tomato invertasegenes), Sogaard et al., J. Biol. Chem. 268: 22480 (1993) (site-directedmutagenesis of barley alpha-amylase gene), and Fisher et al., PlantPhysiol. 102: 1045 (1993) (maize endosperm starch branching enzyme II),WO 99/10498 (improved digestibility and/or starch extraction throughmodification of UDP-D-xylose 4-epimerase, Fragile 1 and 2, Ref1, HCHL,C4H), U.S. Pat. No. 6,232,529 (method of producing high oil seed bymodification of starch levels (AGP)). The fatty acid modification genesmentioned above may also be used to affect starch content and/orcomposition through the interrelationship of the starch and oilpathways.

(D) Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see U.S. Pat. No. 6,787,683,US2004/0034886 and WO 00/68393 involving the manipulation of antioxidantlevels through alteration of a phytl prenyl transferase (ppt), WO03/082899 through alteration of a homogentisate geranyl geranyltransferase (hggt).

(E) Altered essential seed amino acids. For example, see U.S. Pat. No.6,127,600 (method of increasing accumulation of essential amino acids inseeds), U.S. Pat. No. 6,080,913 (binary methods of increasingaccumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389(high lysine), WO99/40209 (alteration of amino acid compositions inseeds), WO99/29882 (methods for altering amino acid content ofproteins), U.S. Pat. No. 5,850,016 (alteration of amino acidcompositions in seeds), WO98/20133 (proteins with enhanced levels ofessential amino acids), U.S. Pat. No. 5,885,802 (high methionine), U.S.Pat. No. 5,885,801 (high threonine), U.S. Pat. No. 6,664,445 (plantamino acid biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increasedlysine and threonine), U.S. Pat. No. 6,441,274 (plant tryptophansynthase beta subunit), U.S. Pat. No. 6,346,403 (methionine metabolicenzymes), U.S. Pat. No. 5,939,599 (high sulfur), U.S. Pat. No. 5,912,414(increased methionine), WO98/56935 (plant amino acid biosyntheticenzymes), WO98/45458 (engineered seed protein having higher percentageof essential amino acids), WO98/42831 (increased lysine), U.S. Pat. No.5,633,436 (increasing sulfur amino acid content), U.S. Pat. No.5,559,223 (synthetic storage proteins with defined structure containingprogrammable levels of essential amino acids for improvement of thenutritional value of plants), WO96/01905 (increased threonine),WO95/15392 (increased lysine), US2003/0163838, US2003/0150014,US2004/0068767, U.S. Pat. No. 6,803,498, WO01/79516, and WO00/09706 (CesA: cellulose synthase), U.S. Pat. No. 6,194,638 (hemicellulose), U.S.Pat. No. 6,399,859 and US2004/0025203 (UDPGdH), U.S. Pat. No. 6,194,638(RGP).

4. Genes that Control Male-Sterility

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

(A) Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT (WO 01/29237).

(B) Introduction of various stamen-specific promoters (WO 92/13956, WO92/13957).

(C) Introduction of the barnase and the barstar gene (Paul et al. PlantMol. Biol. 19:611-622, 1992).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341; 6,297,426; 5,478,369;5,824,524; 5,850,014; and 6,265,640; all of which are herebyincorporated by reference.

5. Genes that create a site for site specific DNA integration. Thisincludes the introduction of FRT sites that may be used in the FLP/FRTsystem and/or Lox sites that may be used in the Cre/Loxp system. Forexample, see Lyznik, et al., Site-Specific Recombination for GeneticEngineering in Plants, Plant Cell Rep (2003) 21:925-932 and WO 99/25821,which are hereby incorporated by reference. Other systems that may beused include the Gin recombinase of phage Mu (Maeser et al., 1991; VickiChandler, The Maize Handbook ch. 118 (Springer-Verlag 1994), the Pinrecombinase of E. coli (Enomoto et al., 1983), and the R/RS system ofthe pSR1 plasmid (Araki et al., 1992).

6. Genes that affect abiotic stress resistance (including but notlimited to flowering, ear and seed development, enhancement of nitrogenutilization efficiency, altered nitrogen responsiveness, droughtresistance or tolerance, cold resistance or tolerance, and saltresistance or tolerance) and increased yield under stress. For example,see: WO 00/73475 where water use efficiency is altered throughalteration of malate; U.S. Pat. No. 5,892,009, U.S. Pat. No. 5,965,705,U.S. Pat. No. 5,929,305,U.S. Pat. No. 5,891,859, U.S. Pat. No.6,417,428, U.S. Pat. No. 6,664,446, U.S. Pat. No. 6,706,866, U.S. Pat.No. 6,717,034, WO2000060089, WO2001026459, WO2001035725, WO2001034726,WO2001035727, WO2001036444, WO2001036597, WO2001036598, WO2002015675,WO2002017430, WO2002077185, WO2002079403, WO2003013227, WO2003013228,WO2003014327, WO2004031349, WO2004076638, WO9809521, and WO9938977describing genes, including CBF genes and transcription factorseffective in mitigating the negative effects of freezing, high salinity,and drought on plants, as well as conferring other positive effects onplant phenotype; US2004/0148654 and WO01/36596 where abscisic acid isaltered in plants resulting in improved plant phenotype such asincreased yield and/or increased tolerance to abiotic stress;WO2000/006341, WO04/090143, U.S. application Ser. Nos. 10/817483 andSer. No. 09/545,334 where cytokinin expression is modified resulting inplants with increased stress tolerance, such as drought tolerance,and/or increased yield. Also see WO0202776, WO2003052063, JP2002281975,U.S. Pat. No. 6,084,153, WO0164898, U.S. Pat. No. 6,177,275, and U.S.Pat. No. 6,107,547 (enhancement of nitrogen utilization and alterednitrogen responsiveness). For ethylene alteration, see US20040128719,US20030166197 and WO200032761. For plant transcription factors ortranscriptional regulators of abiotic stress, see e.g. US20040098764 orUS20040078852.

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants, see e.g.WO97/49811 (LHY), WO98/56918 (ESD4), WO97/10339 and U.S. Pat. No.6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO96/14414 (CON),WO96/38560, WO01/21822 (VRN1), WO00/44918 (VRN2), WO99/49064 (GI),WO00/46358 (FRI), WO97/29123, U.S. Pat. No. 6,794,560, U.S. Pat. No.6,307,126 (GAI), WO99/09174 (D8 and Rht), and WO2004076638 andWO2004031349 (transcription factors).

Using PHWWE to Develop Other Maize Inbreds

Inbred lines such as PHWWE provide a source of breeding material thatmay be used to develop new maize inbred lines. Plant breeding techniquesknown in the art and used in a maize plant breeding program include, butare not limited to, recurrent selection, mass selection, bulk selection,backcrossing, pedigree breeding, open pollination breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection, making double haploids, and transformation. Oftencombinations of these techniques are used. The development of maizehybrids in a maize plant breeding program requires, in general, thedevelopment of homozygous inbred lines, the crossing of these lines, andthe evaluation of the crosses. There are many analytical methodsavailable to evaluate the result of a cross. The oldest and mosttraditional method of analysis is the observation of phenotypic traitsbut genotypic analysis may also be used.

Using PHWWE in a Breeding Program

This invention is directed to methods for producing a maize plant bycrossing a first parent maize plant with a second parent maize plantwherein either the first or second parent maize plant is an inbred maizeplant of the line PHWWE. The other parent may be any other maize plant,such as another inbred line or a plant that is part of a synthetic ornatural population. Any such methods using the inbred maize line PHWWEare part of this invention: selfing, sibbing, backcrosses, massselection, pedigree breeding, bulk selection, hybrid production, crossesto populations, and the like. These methods are well known in the artand some of the more commonly used breeding methods are described below.Descriptions of breeding methods can also be found in one of severalreference books (e.g., Allard, Principles of Plant Breeding, 1960;Simmonds, Principles of Crop Improvement, 1979; Fehr, “Breeding Methodsfor Cultivar Development”, Production and Uses, 2^(nd) ed., Wilcoxeditor, 1987 the disclosure of which is incorporated herein byreference).

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such asPHWWE and one other elite inbred line having one or more desirablecharacteristics that is lacking or which complements PHWWE. If the twooriginal parents do not provide all the desired characteristics, othersources can be included in the breeding population. In the pedigreemethod, superior plants are selfed and selected in successive filialgenerations. In the succeeding filial generations the heterozygouscondition gives way to homogeneous lines as a result of self-pollinationand selection. Typically in the pedigree method of breeding, five ormore successive filial generations of selfing and selection ispracticed: F1→F2; F2→F3; F3→F4; F4→F5, etc. After a sufficient amount ofinbreeding, successive filial generations will serve to increase seed ofthe developed inbred. Preferably, the inbred line comprises homozygousalleles at about 95% or more of its loci.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding to modify PHWWEand a hybrid that is made using the modified PHWWE. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one line, the donor parent, to aninbred called the recurrent parent, which has overall good agronomiccharacteristics yet lacks that desirable trait or traits. However, thesame procedure can be used to move the progeny toward the genotype ofthe recurrent parent but at the same time retain many components of thenon-recurrent parent by stopping the backcrossing at an early stage andproceeding with selfing and selection.

Therefore, an embodiment of this invention is a method of making abackcross conversion of line PHWWE, comprising the steps of crossing aplant of line PHWWE with a donor plant comprising a mutant gene ortransgene conferring a desired trait, selecting an F1 progeny plantcomprising the mutant gene or transgene conferring the desired trait,and backcrossing the selected F1 progeny plant to a plant of maizeinbred line PHWWE. This method may further comprise the step ofobtaining a molecular marker profile of maize inbred line PHWWE andusing the molecular marker profile to select for a progeny plant withthe desired trait and the molecular marker profile of PHWWE. In the samemanner, this method may be used to produce an F1 hybrid seed by adding afinal step of crossing the desired trait conversion of maize inbred linePHWWE with a different maize plant to make F1 hybrid maize seedcomprising a mutant gene or transgene conferring the desired trait.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. PHWWE is suitable for use in a recurrentselection program. The method entails individual plants crosspollinating with each other to form progeny. The progeny are grown andthe superior progeny selected by any number of selection methods, whichinclude individual plant, half-sib progeny, full-sib progeny, selfedprogeny and topcrossing. The selected progeny are cross pollinated witheach other to form progeny for another population. This population isplanted and again superior plants are selected to cross pollinate witheach other. Recurrent selection is a cyclical process and therefore canbe repeated as many times as desired. The objective of recurrentselection is to improve the traits of a population. The improvedpopulation can then be used as a source of breeding material to obtaininbred lines to be used in hybrids or used as parents for a syntheticcultivar. A synthetic cultivar is the resultant progeny formed by theintercrossing of several selected inbreds.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection seeds fromindividuals are selected based on phenotype and/or genotype. Theseselected seeds are then bulked and used to grow the next generation.Bulk selection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Instead of self pollination, directed pollination could beused as part of the breeding program.

Mutation Breeding

Mutation breeding is one of many methods that could be used to introducenew traits into PHWWE. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g.cobalt 60 or cesium 137), neutrons, (product of nuclear fission byuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (preferably from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethylenamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques, such as backcrossing. Details of mutation breedingcan be found in “Principles of Cultivar Development” Fehr, 1993Macmillan Publishing Company, the disclosure of which is incorporatedherein by reference. In addition, mutations created in other lines maybe used to produce a backcross conversion of PHWWE that comprises suchmutation.

Breeding with Molecular Markers

Molecular markers, which includes markers identified through the use oftechniques such as Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats(SSRs) and Single Nucleotide Polymorphisms (SNPs), may be used in plantbreeding methods utilizing PHWWE.

Isozyme Electrophoresis and RFLPs as discussed in Lee, M., “Inbred Linesof Maize and Their Molecular Markers,” The Maize Handbook,(Springer-Verlag, New York, Inc. 1994, at 423-432), have been widelyused to determine genetic composition. Isozyme Electrophoresis has arelatively low number of available markers and a low number of allelicvariants among maize inbreds. RFLPs allow more discrimination becausethey have a higher degree of allelic variation in maize and a largernumber of markers can be found. Both of these methods have been eclipsedby SSRs as discussed in Smith et al., “An evaluation of the utility ofSSR loci as molecular markers in maize (Zea mays L.): comparisons withdata from RFLPs and pedigree”, Theoretical and Applied Genetics (1997)vol. 95 at 163-173 and by Pejic et al., “Comparative analysis of geneticsimilarity among maize inbreds detected by RFLPs, RAPDs, SSRs, andAFLPs,” Theoretical and Applied Genetics (1998) at 1248-1255incorporated herein by reference. SSR technology is more efficient andpractical to use than RFLPs; more marker loci can be routinely used andmore alleles per marker locus can be found using SSRs in comparison toRFLPs. Single Nucleotide Polymorphisms may also be used to identify theunique genetic composition of the invention and progeny lines retainingthat unique genetic composition. Various molecular marker techniques maybe used in combination to enhance overall resolution.

Maize DNA molecular marker linkage maps have been rapidly constructedand widely implemented in genetic studies. One such study is describedin Boppenmaier, et al., “Comparisons among strains of inbreds forRFLPs”, Maize Genetics Cooperative Newsletter, 65:1991, pg. 90, isincorporated herein by reference.

One use of molecular markers is Quantitative Trait Loci (QTL) mapping.QTL mapping is the use of markers, which are known to be closely linkedto alleles that have measurable effects on a quantitative trait.Selection in the breeding process is based upon the accumulation ofmarkers linked to the positive effecting alleles and/or the eliminationof the markers linked to the negative effecting alleles from the plant'sgenome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select for the genome of the recurrent parent and against thegenome of the donor parent. Using this procedure can minimize the amountof genome from the donor parent that remains in the selected plants. Itcan also be used to reduce the number of crosses back to the recurrentparent needed in a backcrossing program. The use of molecular markers inthe selection process is often called genetic marker enhanced selection.

Production of Double Haploids

The production of double haploids can also be used for the developmentof inbreds in the breeding program. For example, an F1 hybrid for whichPHWWE is a parent can be used to produce double haploid plants. Doublehaploids are produced by the doubling of a set of chromosomes (1N) froma heterozygous plant to produce a completely homozygous individual. Forexample, see Wan et al., “Efficient Production of Doubled Haploid PlantsThrough Colchicine Treatment of Anther-Derived Maize Callus”,Theoretical and Applied Genetics, 77:889-892, 1989 and US2003/0005479.This can be advantageous because the process omits the generations ofselfing needed to obtain a homozygous plant from a heterozygous source.

Haploid induction systems have been developed for various plants toproduce haploid tissues, plants and seeds. The haploid induction systemcan produce haploid plants from any genotype by crossing a selected line(as female) with an inducer line. Such inducer lines for maize includeStock 6 (Coe, 1959, Am. Nat. 93:381-382; Sharkar and Coe, 1966, Genetics54:453-464) RWS (see world wide web sitewww.uni-hohenheim.de/%7Eipspwww/350b/indexe.html#Project3), KEMS(Deimling, Roeber, and Geiger, 1997, Vortr. Pflanzenzuchtg 38:203-224),or KMS and ZMS (Chalyk, Bylich & Chebotar, 1994, MNL 68:47; Chalyk &Chebotar, 2000, Plant Breeding 119:363-364), and indeterminategametophyte (ig) mutation (Kermicle 1969 Science 166:1422-1424). Thedisclosures of which are incorporated herein by reference.

Methods for obtaining haploid plants are also disclosed in Kobayashi, M.et al., Journ. of Heredity 71(1):9-14, 1980, Pollacsek, M., Agronomie(Paris) 12(3):247-251, 1992; Cho-Un-Haing et al., Journ. of Plant Biol.,1996, 39(3):185-188; Verdoodt, L., et al., February 1998, 96(2):294-300;Genetic Manipulation in Plant Breeding, Proceedings InternationalSymposium Organized by EUCARPIA, September 8-13, 1985, Berlin, Germany;Chalyk et al., 1994, Maize Genet Coop. Newsletter 68:47; Chalyk, S. T.,1999, Maize Genet. Coop. Newsletter 73:53-54; Coe, R. H., 1959, Am. Nat.93:381-382; Deimling, S. et al., 1997, Vortr. Pflanzenzuchtg 38:203-204;Kato, A., 1999, J. Hered. 90:276-280; Lashermes, P. et al., 1988, Theor.Appl. Genet. 76:570-572 and 76:405-410; Tyrnov, V. S. et al., 1984,Dokl. Akad. Nauk. SSSR 276:735-738; Zabirova, E. R. et al., 1996,Kukuruza I Sorgo N4, 17-19; Aman, M. A., 1978, Indian J. Genet PlantBreed 38:452-457; Chalyk S. T., 1994, Euphytica 79:13-18; Chase, S. S.,1952, Agron. J. 44:263-267; Coe, E. H., 1959, Am. Nat. 93:381-382; Coe,E. H., and Sarkar, K. R., 1964 J. Hered. 55:231-233; Greenblatt, I. M.and Bock, M., 1967, J. Hered. 58:9-13; Kato, A., 1990, Maize Genet.Coop. Newsletter 65:109-110; Kato, A., 1997, Sex. Plant Reprod.10:96-100; Nanda, D. K. and Chase, S. S., 1966, Crop Sci. 6:213-215;Sarkar, K. R. and Coe, E. H., 1966, Genetics 54:453-464; Sarkar, K. R.and Coe, E. H., 1971, Crop Sci. 11:543-544; Sarkar, K. R. and Sachan J.K. S., 1972, Indian J. Agric. Sci. 42:781-786; Kermicle J. L., 1969,Mehta Yeshwant, M. R., Genetics and Molecular Biology, September 2000,23(3):617-622; Tahir, M. S. et al. Pakistan Journal of Scientific andIndustrial Research, August 2000, 43(4):258-261; Knox, R. E. et al.Plant Breeding, August 2000, 119(4):289-298; U.S. Pat. No. 5,639,951 andU.S. patent application Ser. No. 10/121,200, the disclosures of whichare incorporated herein by reference.

Thus, an embodiment of this invention is a process for making asubstantially homozygous PHWWE progeny plant by producing or obtaining aseed from the cross of PHWWE and another maize plant and applying doublehaploid methods to the F1 seed or F1 plant or to any successive filialgeneration. Such methods decrease the number of generations required toproduce an inbred with similar genetics or characteristics to PHWWE. SeeBernardo, R. and Kahler, A. L., Theor. Appl. Genet. 102:986-992, 2001.

In particular, a process of making seed retaining the molecular markerprofile of maize inbred line PHWWE is contemplated, such processcomprising obtaining or producing F1 hybrid seed for which maize inbredline PHWWE is a parent, inducing doubled haploids to create progenywithout the occurrence of meiotic segregation, obtaining the molecularmarker profile of maize inbred line PHWWE, and selecting progeny thatretain the molecular marker profile of PHWWE.

Use of PHWWE in Tissue Culture

This invention is also directed to the use of PHWWE in tissue culture.As used herein, the term “tissue culture” includes plant protoplasts,plant cell tissue culture, cultured microspores, plant calli, plantclumps, and the like. As used herein, phrases such as “growing the seed”or “grown from the seed” include embryo rescue, isolation of cells fromseed for use in tissue culture, as well as traditional growing methods.

Duncan, Williams, Zehr, and Widholm, Planta (1985) 165:322-332 reflectsthat 97% of the plants cultured that produced callus were capable ofplant regeneration. Subsequent experiments with both inbreds and hybridsproduced 91% regenerable callus that produced plants. In a further studyin 1988, Songstad, Duncan & Widholm in Plant Cell Reports (1988),7:262-265 reports several media additions that enhance regenerability ofcallus of two inbred lines. Other published reports also indicated that“nontraditional” tissues are capable of producing somatic embryogenesisand plant regeneration. K. P. Rao, et al., Maize Genetics CooperationNewsletter, 60:64-65 (1986), refers to somatic embryogenesis from glumecallus cultures and B. V. Conger, et al., Plant Cell Reports, 6:345-347(1987) indicates somatic embryogenesis from the tissue cultures of maizeleaf segments. Thus, it is clear from the literature that the state ofthe art is such that these methods of obtaining plants are, and were,“conventional” in the sense that they are routinely used and have a veryhigh rate of success.

Tissue culture of maize, including tassel/anther culture, is describedin U.S. 2002/0062506A1 and European Patent Application, publicationEP0160,390, each of which are incorporated herein by reference for thispurpose. Maize tissue culture procedures are also described in Green andRhodes, “Plant Regeneration in Tissue Culture of Maize,” Maize forBiological Research “Plant Regeneration in Tissue Cultures of Maize” (INMaize for Biological Research, 1982, pp. 367-372) and in Duncan, et al.,“The Production of Callus Capable of Plant Regeneration from ImmatureEmbryos of Numerous Zea Mays Genotypes,” 165 Planta 322-332 (1985).Thus, another aspect of this invention is to provide cells which upongrowth and differentiation produce maize plants having the genotypeand/or physiological and morphological characteristics of inbred linePHWWE.

Progeny Plants

All plants produced by the use of the methods described herein and thatretain the unique genetic or trait combinations of PHWWE are within thescope of the invention. Progeny of the breeding methods described hereinmay be characterized in any number of ways, such as by traits retainedin the progeny, pedigree and/or molecular markers. Combinations of thesemethods of characterization may be used.

Breeder's of ordinary skill in the art have developed the concept of an“essentially derived variety”, which is defined in 7 U.S.C. §2104(a)(3)of the Plant Variety Protection Act and is hereby incorporated byreference. Varieties and plants that are essentially derived from PHWWEare within the scope of the invention.

Pedigree is a method used by breeders of ordinary skill in the art todescribe the varieties. Varieties that are more closely related bypedigree are likely to share common genotypes and combinations ofphenotypic characteristics. All breeders of ordinary skill in the artmaintain pedigree records of their breeding programs. These pedigreerecords contain a detailed description of the breeding process,including a listing of all parental lines used in the breeding processand information on how such line was used. One embodiment of thisinvention is progeny plants and parts thereof with at least one ancestorthat is PHWWE, and more specifically, where the pedigree of the progenyincludes 1, 2, 3, 4, and/or 5 or less breeding crosses to a maize plantother than PHWWE or a plant that has PHWWE as a parent or otherprogenitor. A breeder of ordinary skill in the art would know if PHWWEwere used in the development of a progeny line, and would also know howmany crosses to a line other than PHWWE or line with PHWWE as a parentor other progenitor were made in the development of any progeny line.

Molecular markers also provide a means by which those of ordinary skillin the art characterize the similarity or differences of two lines.Using the breeding methods described herein, one can develop individualplants, plant cells, and populations of plants that retain at least 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 99.5% genetic contribution from inbred line PHWWE,as measured by either percent identity or percent similarity. On average50% of the starting germplasm would be expected to be passed to theprogeny line after one cross to another line, 25% after another cross toa different line, and so on. With backcrossing, the expectedcontribution of PHWWE after 2, 3, 4 and 5 doses (or 1, 2, 3 and 4backcrosses) would be 75%, 87.5%, 93.75% and 96.875% respectively.Actual genetic contribution may be much higher than the geneticcontribution expected by pedigree, especially if molecular markers areused in selection. Molecular markers could also be used to confirmand/or determine the pedigree of the progeny line.

A breeder will commonly work to combine a specific trait of anundeveloped variety of the species, such as a high level of resistanceto a particular disease, with one or more of the elite agronomiccharacteristics (yield, maturity, plant size, lodging resistance, etc.)needed for use as a commercial variety. This combination, oncedeveloped, provides a valuable source of new germplasm for furtherbreeding. For example, it may take 10-15 years and significant effort toproduce such a combination, yet progeny may be developed that retainthis combination in as little as 2-5 years and with much less effort.

Specific Embodiments

Specific methods and products produced using line PHWWE in plantbreeding are discussed in the following sections. The methods outlinedare described in detail by way of illustration and example for purposesof clarity and understanding. However, it will be obvious that certainchanges and modifications may be practiced within the scope of theinvention.

One method for producing a line derived from inbred line PHWWE is asfollows. One of ordinary skill in the art would produce or obtain a seedfrom the cross between inbred line PHWWE and another variety of maize,such as an elite inbred variety. The F1 seed derived from this crosswould be grown to form a homogeneous population. The F1 seed wouldcontain essentially all of the alleles from variety PHWWE andessentially all of the alleles from the other maize variety. The F1nuclear genome would be made-up of 50% variety PHWWE and 50% of theother elite variety. The F1 seed would be grown and allowed to self,thereby forming F2 seed. On average the F2 seed would have derived 50%of its alleles from variety PHWWE and 50% from the other maize variety,but many individual plants from the population would have a greaterpercentage of their alleles derived from PHWWE (Wang J. and R. Bernardo,2000, Crop Sci. 40:659-665 and Bernardo, R. and A. L. Kahler, 2001,Theor. Appl. Genet 102:986-992). The molecular markers of PHWWE could beused to select and retain those lines with high similarity to PHWWE. TheF2 seed would be grown and selection of plants would be made based onvisual observation, markers and/or measurement of traits. The traitsused for selection may be any PHWWE trait described in thisspecification, including the inbred per se maize PHWWE traits describedherein under the detailed description of inbred PHWWE. Such traits mayalso be the good general or specific combining ability of PHWWE,including its ability to produce hybrids with the approximate maturityand/or hybrid combination traits described herein under the detaileddescription of inbred PHWWE. The PHWWE progeny plants that exhibit oneor more of the desired PHWWE traits, such as those listed herein, wouldbe selected and each plant would be harvested separately. This F3 seedfrom each plant would be grown in individual rows and allowed to self.Then selected rows or plants from the rows would be harvestedindividually. The selections would again be based on visual observation,markers and/or measurements for desirable traits of the plants, such asone or more of the desirable PHWWE traits listed herein. The process ofgrowing and selection would be repeated any number of times until aPHWWE progeny inbred plant is obtained. The PHWWE progeny inbred plantwould contain desirable traits derived from inbred plant PHWWE, some ofwhich may not have been expressed by the other maize variety to whichinbred line PHWWE was crossed and some of which may have been expressedby both maize varieties but now would be at a level equal to or greaterthan the level expressed in inbred variety PHWWE. However, in each casethe resulting progeny line would benefit from the efforts of theinventor(s), and would not have existed but for the inventor(s) work increating PHWWE. The PHWWE progeny inbred plants would have, on average,50% of their nuclear genes derived from inbred line PHWWE, but manyindividual plants from the population would have a greater percentage oftheir alleles derived from PHWWE. This breeding cycle, of crossing andselfing, and optional selection, may be repeated to produce anotherpopulation of PHWWE progeny maize plants with, on average, 25% of theirnuclear genes derived from inbred line PHWWE, but, again, manyindividual plants from the population would have a greater percentage oftheir alleles derived from PHWWE. This process can be repeated for athird, fourth, fifth, sixth, seventh or more breeding cycles. Anotherembodiment of the invention is a PHWWE progeny plant that has receivedthe desirable PHWWE traits listed herein through the use of PHWWE, whichtraits were not exhibited by other plants used in the breeding process.

Therefore, an embodiment of this invention is a PHWWE progeny maizeplant, wherein at least one ancestor of said PHWWE progeny maize plantis the maize plant or plant part of PHWWE, and wherein the pedigree ofsaid PHWWE progeny maize plant is within two breeding crosses of PHWWEor a plant that has PHWWE as a parent. The progeny plants, parts andplant cells produced from PHWWE may be further characterized as having apercent marker similarity or identity with PHWWE as described herein.

The previous example can be modified in numerous ways, for instanceselection may or may not occur at every selfing generation, selectionmay occur before or after the actual self-pollination process occurs, orindividual selections may be made by harvesting individual ears, plants,rows or plots at any point during the breeding process described. Doublehaploid breeding methods may be used at any step in the process. Insteadof selfing out of the hybrid produced from the inbred, one could firstcross the hybrid to either a parent line or a different inbred, and thenself out of that cross.

The population of plants produced at each and any cycle of breeding isalso an embodiment of the invention, and on average each such populationwould predictably consist of plants containing approximately 50% of itsgenes from maize line PHWWE in the first breeding cycle, 25% of itsgenes from maize line PHWWE in the second breeding cycle, 12.5% of itsgenes from inbred line PHWWE in the third breeding cycle, 6.25% in thefourth breeding cycle, 3.125% in the fifth breeding cycle, and so on.However, in each case the use of PHWWE provides a substantial benefit.The linkage groups of PHWWE would be retained in the progeny lines, andsince current estimates of the maize genome size is about 50,000-80,000genes (Xiaowu, Gai et al., Nucleic Acids Research, 2000, Vol. 28, No. 1,94-96), in addition to non-coding DNA that impacts gene expression, itprovides a significant advantage to use PHWWE as starting material toproduce a line that retains desired genetics or traits of PHWWE.

Therefore, an embodiment of the invention is a process for making apopulation of PHWWE progeny inbred maize plants comprising obtaining orproducing a first generation progeny maize seed comprising the plant ofPHWWE as a parent, growing said first generation progeny maize seed toproduce first generation maize plants and obtaining self or sibpollinated seed from said first generation maize plants, and growing theself or sib pollinated seed to obtain a population of PHWWE progenyinbred maize plants.

The population of PHWWE progeny inbred maize plants produced by thismethod are also embodiments of the invention, and such population as awhole will retain the expected genetic contribution of PHWWE. An inbredline selected from the population of PHWWE progeny inbred maize plantsproduced by this method is an embodiment, and such line may be furthercharacterized by its molecular marker identity or similarity to PHWWE.

In this manner, the invention also encompasses a process for making aPHWWE inbred progeny maize plant comprising the steps of obtaining orproducing a first generation progeny maize seed wherein a parent of saidfirst generation progeny maize seed is a PHWWE plant, growing said firstgeneration progeny maize seed to produce a first generation maize plantand obtaining self or sib pollinated seed from said first generationmaize plant, and producing successive filial generations to obtain aPHWWE inbred progeny maize plant. Also an embodiment of this inventionis the first breeding cycle inbred PHWWE maize plant produced by thismethod.

Crosses to Other Species

The utility of inbred maize line PHWWE also extends to crosses withother species. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Coix,Schlerachne, Polytoca, Chionachne, and Trilobachne, of the tribeMaydeae. Potentially suitable for crosses with PHWWE may be the variousvarieties of grain sorghum, Sorghum bicolor (L.) Moench.

Deposits

Applicant will make a deposit of at least 2500 seeds of Inbred MaizeLine PHWWE with the American Type Culture Collection (ATCC), Manassas,Va. 20110 USA, ATCC Deposit No. ______ The seeds to be deposited withthe ATCC on ______ will be taken from the deposit maintained by PioneerHi-Bred International, Inc., 7250 NW 62^(nd) Avenue, Johnston, Iowa,50131 since prior to the filing date of this application. Access to thisdeposit will be available during the pendency of the application to theCommissioner of Patents and Trademarks and persons determined by theCommissioner to be entitled thereto upon request. Upon allowance of anyclaims in the application, the Applicant will make the deposit availableto the public pursuant to 37 C.F.R. §1.808. This deposit of the InbredMaize Line PHWWE will be maintained in the ATCC depository, which is apublic depository, for a period of 30 years, or 5 years after the mostrecent request, or for the enforceable life of the patent, whichever islonger, and will be replaced if it becomes nonviable during that period.Additionally, Applicant has or will satisfy all of the requirements of37 C.F.R. §§1.801-1.809, including providing an indication of theviability of the sample upon deposit. Applicant has no authority towaive any restrictions imposed by law on the transfer of biologicalmaterial or its transportation in commerce. Applicant does not waive anyinfringement of rights granted under this patent or under the PlantVariety Protection Act (7 USC 2321 et seq.). U.S. Plant VarietyProtection of Inbred Maize Line PHWWE has been applied for. Unauthorizedseed multiplication prohibited.

TABLE 3 SSR PROFILE DATA FOR PHWWE Marker Base Bin Name Pairs 1.00umc1041 327 1.00 umc1354 309.65 1.01 phi056 255.3 1.01 umc1071 117 1.01umc1177 107.7 1.01 umc1269 344.475 1.01 umc1484 211.5 1.01 umc201273.825 1.01 umc2224 354.695 1.03 umc1701 117.675 1.04 umc1452 360.9 1.04umc2112 311.5 1.04 umc2217 163.75 1.05 umc1244 348.275 1.05 umc1297159.85 1.05 umc1689 149.5 1.05 umc1734 251 1.05 umc2025 131.35 1.06umc1396 169.1 1.06 umc1508 246.5 1.06 umc1668 146.25 1.06 umc1709 350.651.06 umc1754 224.9 1.06 umc1924 161.35 1.06 umc2234 150.5 1.07 phi00273.53 1.07 umc1128 226.9 1.07 umc1245 305.4 1.07 umc1833 136.3 1.07umc2237 162.05 1.08 umc1446 161.3 1.08 umc2385 264.35 1.09 umc1298362.65 1.09 umc1715 152.5 1.09 umc2047 133.25 1.10 umc1885 145.875 1.10umc2149 152.375 1.11 umc1553 276 1.11 umc1737 350.5 1.11 umc1862 143.051.11 umc2242 382 2.00 umc1419 106.7 2.00 umc2245 150.1 2.02 umc1518222.5 2.03 bnlg1621 188 2.04 phi083 125.56 2.04 umc1024 326.05 2.04umc1026 123.95 2.04 umc1410 214.175 2.04 umc1465 394.75 2.04 umc1541320.525 2.04 umc2030 168.5 2.04 umc2125 138.15 2.04 umc2247 254.6 2.04umc2248 154.125 2.05 umc1459 95.45 2.06 umc1658 142.1 2.06 umc1749 206.12.06 umc1875 146 2.06 umc2023 146.925 2.06 umc2192 335 2.06 umc2254105.95 2.07 umc1108 205.3 2.07 umc1554 326.825 2.07 umc1637 120.6 2.07umc2205 174.95 2.07 umc2374 263 2.08 phi090 146.005 2.08 umc1230 310.12.08 umc1526 105 2.08 umc1745 216 2.09 umc1551 240.75 3.00 umc2118 319.33.01 umc1394 244.3 3.01 umc2071 150.5 3.01 umc2256 165.5 3.01 umc2376149.5 3.02 umc1458 335.15 3.04 umc1030 240 3.04 umc1347 228.35 3.04umc1392 148.7 3.04 umc1495 105.6 3.04 umc1908 133.6 3.04 umc2002 125.7253.04 umc2117 355.75 3.04 umc2263 393.4 3.05 phi053 166.74 3.05 phi073187.785 3.05 umc1307 134.05 3.05 umc1400 464.6 3.05 umc2265 203.275 3.06umc1027 201.05 3.06 umc1311 212 3.06 umc1644 154.95 3.06 umc1949 112.2253.06 umc1985 257.875 3.06 umc2270 139.85 3.07 umc1286 234.05 3.07umc1528 120.875 3.07 umc1690 166.5 3.07 umc1825 160.1 3.07 umc2273233.95 3.08 umc1273 205.825 3.08 umc1844 142.75 3.08 umc2276 135.2 4.01phi072 139.43 4.04 mmc0471a 222 4.05 umc1317 113.8 4.05 umc1390 133.54.05 umc1451 109.05 4.05 umc1791 153.425 4.05 umc1851 138.5 4.05 umc1895147.875 4.05 umc2061 137.35 4.06 bnlg2291 178.925 4.06 bnlg252 165.9254.06 mmc0371 275 4.06 umc1702 95 4.06 umc1869 151.5 4.06 umc1945 113.54.06 umc2027 116.525 4.07 umc1620 148.35 4.07 umc1651 95.625 4.07umc1847 160.15 4.08 bnlg1927 198.9 4.08 umc1051 125.9 4.08 umc1132 132.54.08 umc1559 141.35 4.08 umc1667 147 4.08 umc1856 156.9 4.08 umc1871135.5 4.09 umc1101 137.6 4.09 umc1650 137 4.09 umc1740 98.35 4.09umc1834 163.425 4.09 umc1940 128.5 4.09 umc1999 125.8 4.09 umc2046 115.84.09 umc2139 138.775 5.00 umc1097 109.525 5.00 umc1445 225.1 5.00umc1491 248.275 5.00 umc2022 153.5 5.00 umc2292 137.675 5.01 phi024361.6 5.01 umc1365 115.05 5.01 umc1894 159.325 5.02 umc1587 143.6 5.03umc1355 357.85 5.03 umc1731 364.7 5.03 umc1830 196.35 5.03 umc2297 1515.03 umc2400 211.6 5.04 umc1060 231.075 5.04 umc1221 148.35 5.04 umc1332205.75 5.04 umc1629 114.5 5.04 umc1815 274.5 5.04 umc1990 132.75 5.04umc2302 348.45 5.05 umc1348 226 5.05 umc1482 216.1 5.05 umc1800 154.155.05 umc1822 103 5.06 phi085 233.635 5.06 umc1941 122 5.06 umc2198166.25 5.06 umc2305 164.35 5.07 umc2013 131.4 5.08 umc1225 109.75 5.08umc1792 120.725 5.09 umc1153 105.225 5.09 umc2209 167.8 6.00 umc1002123.3 6.00 umc1018 349.7 6.00 umc1883 86.175 6.01 phi077 125 6.01umc1186 268.675 6.01 umc1195 138.175 6.02 umc1006 223.675 6.02 umc1572209.1 6.02 umc1628 124.725 6.02 umc1656 136.775 6.04 umc1014 313.05 6.04umc1614 335.625 6.05 umc1020 146.5 6.05 umc1352 149 6.06 umc1424 293.956.07 phi070 78.235 6.07 umc1350 123 6.07 umc1490 258.5 6.07 umc1621209.6 6.07 umc1653 244.475 6.08 umc2059 147.875 7.00 umc1241 121.25 7.00umc1642 153.4 7.02 umc1068 341 7.02 umc1393 259.5 7.02 umc1401 165.77.02 umc2057 156.075 7.03 umc1841 109.15 7.03 umc1001 145.25 7.03umc1134 321.225 7.03 umc1275 314.1 7.03 umc1324 212.175 7.03 umc1450130.35 7.03 umc1456 128 7.03 umc1567 323.2 7.03 umc1865 151.8 7.04umc1342 231.45 7.04 umc1710 246.355 7.04 umc1799 104.55 7.05 umc1154261.15 7.05 umc1760 224.3 7.06 phi116 165.04 8.01 umc1075 243.875 8.01umc1483 310.75 8.01 umc1786 353.7 8.02 umc1304 251.5 8.02 umc1790 153.58.02 umc1872 148.5 8.02 umc1974 485.7 8.02 umc2004 95.675 8.03 phi115302.625 8.03 umc1034 137 8.03 umc1457 341.125 8.03 umc1470 348.9 8.03umc1741 160.95 8.03 umc1910 161.25 8.05 umc1562 239.7 8.08 phi015100.105 8.09 umc1638 141 9.01 umc1588 323 9.01 umc1596 106.45 9.01umc1809 230.325 9.01 umc2362 167.55 9.02 umc1170 241.025 9.02 umc1636181.7 9.02 umc2336 258.4 9.03 bnlg127 222.5 9.03 phi022 240.55 9.03umc1420 316.95 9.03 umc1691 142 9.03 umc1743 134 9.03 umc2087 266.259.03 umc2337 139.35 9.03 umc2370 133.4 9.04 umc1267 342.275 9.04 umc1522252.95 9.04 umc2394 366.35 9.04 umc2398 126.25 9.05 umc1357 251 9.05umc1519 220.25 9.05 umc1657 164.35 9.05 umc2341 130.3 9.05 umc2371 151.69.06 umc2346 300.5 9.07 bnlg1375 117.75 9.07 umc1104 216.925 9.07umc1505 142.175 9.07 umc2089 137.5 10.00 umc1293 161.275 10.01 umc1318216.5 10.01 umc2053 100.8 10.02 umc1432 119.05 10.02 umc1582 274.5 10.02umc2034 132.55 10.02 umc2069 374.95 10.03 umc1345 166.5 10.03 umc1785218 10.03 umc1938 154.5 10.03 umc2067 152 10.04 phi062 157.805 10.04umc1272 206.5 10.04 umc1280 432.225 10.04 umc1330 340.275 10.04 umc1648144 10.04 umc1678 154.5 10.04 umc1930 102.6 10.04 umc2003 96.4 10.05umc1506 168.65 10.06 umc1249 242 10.06 umc1993 108.7 10.07 umc1176 348.510.07 umc1344 210.755 10.07 umc1569 234.575 10.07 umc1640 103.925 10.07umc1645 165.8 10.07 umc2021 135.5

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All such publications, patents and patentapplications are incorporated by reference herein for the purpose citedto the same extent as if each was specifically and individuallyindicated to be incorporated by reference herein.

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding. Asis readily apparent to one skilled in the art, the foregoing are onlysome of the methods and compositions that illustrate the embodiments ofthe foregoing invention. It will be apparent to those of ordinary skillin the art that variations, changes, modifications and alterations maybe applied to the compositions and/or methods described herein withoutdeparting from the true spirit, concept and scope of the invention.

1. A seed of maize line designated PHWWE, representative seed of saidline having been deposited under ATCC Accession number PTA-XXXX.
 2. Amaize plant, or a part thereof, produced by growing the seed of claim 1.3. Pollen of the plant of claim
 2. 4. An ovule or ovules of the plant ofclaim
 2. 5. A maize cell from the maize plant of claim
 2. 6. The processof transforming the maize cell of claim
 5. 7. Protoplast produced fromthe maize plant cell of claim
 5. 8. A plant according to claim 2,wherein said plant is modified by the addition of at least one mutant ortransgenic gene that confers a characteristic selected from the groupconsisting of male sterility, herbicide resistance, increasedtransformability, increased culturability, a colored marker, aninducible marker, site-specific recombination, disease resistance,insect resistance, altered phosphorus, altered antioxidants, alteredfatty acids, altered essential amino acids, altered carbohydrates andabiotic stress tolerance.
 9. The maize plant of claim 8, wherein saidsite-specific recombination is conferred by a member of the groupconsisting of flp/frt, cre/lox, Gin, Pin, and R/RS.
 10. A process forproducing an F1 hybrid maize seed, said process comprising crossing theplant of claim 2 with a different maize plant and harvesting F1 hybridmaize seed.
 11. The process of claim 10, further comprising growing theF1 hybrid maize seed to produce a hybrid maize plant.
 12. The process ofclaim 11, further comprising culturing a cell from the F1 hybrid maizeseed.
 13. A maize plant cell comprising 95% of the alleles of line PHWWEat the SSR loci listed in Table 3, representative seed of said linehaving been deposited under ATCC Accession number PTA-XXXX.
 14. Themaize plant cell of claim 13 comprising 98% of said alleles.
 15. Themaize plant cell of claim 13 comprising 99% of said alleles.
 16. Themaize plant cell of claim 13 comprising 100% of said alleles.
 17. Theprocess of transforming the maize plant cell of claim
 13. 18. A processof producing an F1 cell comprising crossing a PHWWE plant grown fromPHWWE seed, representative seed of which has been deposited under ATCCAccession number PTA-XXXX, with of another maize line to produce an F1cell.
 19. The F1 cell produced by claim 18.